Human granulocyte specific nuclear antigen(s). I: Production of antisera and determination of specificity.

Non-histone protein-DNA complexes isolated from human lung tissue were used to immunize New Zealand rabbits. Quantitative microcomplement fixation and immunocytochemistry revealed a specific chromatin antigen(s) in the human granulocyte nucleus. The study demonstrates the feasibility of employing antisera against chromosomal non-histone protein-DNA complexes for the immunochemical identification of cell types.     

Analysis of excitation-contraction-coupling components in chronically stimulated canine skeletal muscle.

The chronic stimulation of predominantly fast-twitch mammalian skeletal muscle causes a transformation to physiological characteristics of slow-twitch skeletal muscle. Here, we report the effects of chronic stimulation on the protein components of the sarcoplasmic reticulum and transverse tubular membranes which are directly involved in excitation-contraction coupling. Comparison of protein composition of microsomal fractions from control and chronically stimulated muscle was performed by immunoblot analysis and also by staining with Coomassie blue or the cationic carbocyanine dye Stains-all. Consistent with previous experiments, a greatly reduced density was observed for the fast-twitch isozyme of Ca(2+)-ATPase, while the expression of the slow-twitch Ca(2+)-ATPase was found to be greatly enhanced. Components of the sarcolemma (Na+/K(+)-ATPase, dystrophin-glycoprotein complex) and the free sarcoplasmic reticulum (Ca(2+)-binding protein sarcalumenin and a 53-kDa glycoprotein) were not affected by chronic stimulation. The relative abundance of calsequestrin was slightly reduced in transformed skeletal muscle. However, the expression of the ryanodine receptor/Ca(Ca2+)-release channel from junctional sarcoplasmic reticulum and the transverse tubular dihydropyridine-sensitive Ca2+ channel, as well as two junctional sarcoplasmic reticulum proteins of 90 kDa and 94 kDa, was greatly suppressed in transformed muscle. Thus, the expression of the major protein components of the triad junction involved in excitation-contraction coupling is suppressed, while the expression of other muscle membrane proteins is not affected in chronically stimulated muscle.     

Left superior vena cava with atrial displacement.

Two cadavers were dissected showing persistence of left-sided superior vena cava with atrial displacement and inversion of the venous drainage from the body wall (azygos system of veins). The presence of atrial displacement in relation to a persistent left superior vena cava observed in this study lends support to the view that haemodynamic factors influence the morphogenesis and development of the heart. The development of an asymmetrical venous pattern from a symmetrical venous drainage may be influenced by the same factors which influence the arterial end of the primitive heart tube.     

INTRACELLULAR PHYTOCHROME DISTRIBUTION AS A FUNCTION OF ITS MOLECULAR FORM AND OF ITS DESTRUCTION

The immunocytochemically observed intracellular redistribution of phytochrome as a function of its molecular form is described by utilizing color photomicrography. The reversible change from a diffuse to a discretely localized distribution following photoconversion of the red-absorbing Pr form to the far-red-absorbing Pfr form observed with etiolated oat (Avena sativa L., cv. Garry) coleoptile parenchyma cells is not seen with etiolated wheat (Triticum sativum L., cv. unknown), barley (Hordeum vulgare L., cv. Harrison), or rye (Secale cereale L., cv. Balbo). Whether redistribution in these latter cases does not occur or is below the limit of detection is not known. Upon continuous actinic irradiation, phytochrome, which is discretely localized as Pfr, rapidly disappears by both immunocytochemical and spectral assay. However, after about 90 min irradiation, a new association of phytochrome with nuclei is evident which is more pronounced after 4 or 8 h of irradiation. With longer irradiation times there is a total loss of antigenically detectable phytochrome at the resolution employed in these experiments.     

Comparison of the bile salts of frogs with those of their tadpoles. Bile-salt changes during the metamorphosis of Rana Catesbeiana Shaw.

1. The bile salts of three frog species of the genus Ptychadena and of Rana catesbeiana have been compared with those of their tadpoles. For R. catesbeiana comparison was made of the bile salts in at least ten of the recognized stages of tadpole metamorphosis. 2. In all cases, adult bile salts were more complex than those of the tadpoles. 3. In R. catesbeiana after stage 18, 26-deoxy-5 alpha-ranol was hydroxylated to form 5 alpha-ranol (27-nor-5 alpha-cholestane-3 alpha, 7 alpha, 12 alpha, 24 xi, 26-pentol) and at least two other bile alcohols appeared in solvolysed bile salts. 4. Tadpole bile salts were not found to be biochemically more primitive than those of fully metamorphosed frogs; in some, but not all, cases tadpole bile alcohols could be regarded as biochemical precursors of those in the adult frogs. 5. Detailed evidence for the structure of the bile salts from mass-spectral fragmentation patterns has been deposited as Supplementary Publication SUP 50097 (2 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.     

Cross-linking studies on a cytochrome c-cytochrome c oxidase complex.

A cytochrome $\text{c}$ - cytochrome $\text{c}$ oxidase complex containing 0.8–1.0 moles of cytochrome $\text{c}$ per mole of cytochrome $\text{c}$ oxidase (heme a + a₃) was isolated as described by Ferguson-Miller, S., Brautigan, D.L., and Margoliash E., J. Biol. Chem. $\text{251}$, 1104 (1976). This complex was reacted with dithiobissuccinimidyl propionate, an 11 Å bridging bifunctional reagent, and the cross-linked products obtained were analyzed by two dimensional gel electrophoresis. Cytochrome $\text{c}$ was cross-linked to subunit II of cytochrome $\text{c}$ oxidase. Other cross-linked products were formed involving different subunits of cytochrome $\text{c}$ oxidase. These included I+V, II+V, III+V, V+VII, IV+VI and IV+VII. Experiments are also described using N,N′-bis(3-succinimidyloxycarbonylpropyl) tartarate. The major product formed with this 18 Å bridging bifunctional reagent was a pair containing II+VI.     

Further characterization of human erythrocyte superoxide dismutase.

1. A simplified procedure for the preparation of highly purified human superoxide dismutase from erythrocytes was developed which avoided extremes of pH and ionic strength and the use of organic solvents; the properties of human and bovine proteins, prepared by the method, were compared. 2. Using the two dimensional electrophoretic procedure of O'Farrell, the human superoxide dismutase was found to consist of a single type of polypeptide. 3. The human protein was found to have a total of eight half-cystine residues per mole of protein, compared to six such residues for the bovine protein. The human protein has two sulfhydryl groups which are reactive toward mercurials when dissolved in 1M guanidine-hydrochloride and approximately 3 reactive sulfhydrls when the protein is dissolved in 6 M guanidine hydrochloride. The distribution of the eight sulfur atoms appears to consist of four involved in disulfide linkages, two deeply buried within the molecule and unreactive except under strongly denaturing conditions, and two which are reactive under mildly denaturing conditions. No zero-valent sulfur was found. 4. The visible optical absorption, the visible circular dichroism, and the electron paramagnetic resonance spectra are essentially identical with those of the bovine protein. No unusual absorbance was found at 330 nm. The near ultraviolet spectrum is different from that of the bovine protein, and this appears to be due to differing amino acid compositions. 5. Two fractions of superoxide dismutase activity were observed during chromatography of partially purified solutions on diethylaminoethyl-cellulose. The minor, less mobile form, was found to revert to the less mobile species on aging; the reverse process was not observed to occur. The minor component was found to contain equimolar amounts of Zn and Cu and to have a specific dismutase activity somewhat higher than that of the purified major fraction.